Microbubbles—comprising low solubility gas bubbles (less than 10 μm in diameter) stabilized with a shell (lipid, protein, or polymer) can be used as an ultrasound contrast agent. Targeted microbubbles have been fabricated by incorporating a microbubble shell with ligands specific to molecular markers (e.g. ICAM-1 and P-selectin for cardiovascular-related diseases). These ligands have been shown to allow microbubbles to attach to specific regions of a vascular endothelium through the specific ligand-receptor bond, thereby enabling applications for both targeted molecular imaging and targeted gene/drug delivery. In order to increase the binding efficacy of targeted microbubbles to potential binding sites, especially in large blood vessel environments, acoustic radiation force (ARF) can be applied. In addition, targeted microbubbles incorporated with multiple ligands can be used to further increase adhesion to the vessel wall.
The detection and enhancement of signals derived from ligand-receptor bound microbubbles (“specifically bound adherent microbubbles”) and suppression of surrounding tissue and freely circulating microbubbles can be a central technical challenge in ultrasound-based targeted molecular imaging. Nonlinear signal detection methods (e.g. pulse inversion or harmonic imaging) have been used to eliminate signals from surrounding tissue. Thereafter, signals from “free” microbubbles have been suppressed by lengthy waiting periods (e.g. 15-30 min) to clear the vessel lumen, or by low-pass interframe filtering from recently developed real-time targeted molecular imaging techniques. Previous approaches are only capable of detecting adherent microbubbles and cannot distinguish between non-specific molecular binding (undesirable “signal”), which increases with applied ARF, and specific binding (desirable “signal”). Therefore, all of the previously described techniques require control groups using deactivated microbubbles (or a non-activated flow phantom in an in vitro test) to estimate the non-specific adhesion “background” signal so that a true picture of specifically bound microbubbles can be found.
The presence of the targeted molecular entity along a vascular wall can be assumed if there is a significant increase in adherent microbubbles between control and targeted groups. A consequence of having to use a control group in addition to a test group is that multiple microbubble populations may need to be used resulting in very long procedure times, up to 30-40 minutes, as it often requires at least 20 minutes for microbubbles to clear the vasculature after a single injection. In addition, the specificity of the detection of molecular targets can be limited due to detection of an undesired positive signal from control groups (the control group microbubble signal is often 20% or more of targeted group signal).